Capacity Factor in HPLC: How to understand

Table of Contents

• Introduction and Background
• Capacity Factor
• Calculation
• Controlling factors
• Acceptance criteria
• Case study
• Applications
• Conclusion

Introduction and Background; Capacity Factor in HPLC

The capacity factor plays an important role during chromatographic separation in HPLC.This article will clear all your doubts related to capacity factor and you will be able to replicate it during method development. After reading this article you will know:

• What is the capacity factor?
• How capacity is calculated?
• How capacity is important for chromatographic method development
• What are the factors affecting the capacity factor?
• What are the acceptance criteria for the capacity factor?

Capacity Factor

The capacity factor is a measure of where the peak of interest is located relative to the void volume peak or unretained peak. It is also called the Retention factor. It indicates whether an analyte is retained or not in the experimental chromatographic condition. It depends upon several factors. It is denoted by K.

Capacity Factor Formula

The Following formula is widely used in the Pharmaceutical industries to calculate the Capacity factor:

Where:

• K = Capacity factor
• t_R = Retention time of concerned analyte
• t_o = Retention time of unretained peak or Void volume peak

Controlling factors

• Structure/nature of the molecules: The capacity factor depends upon the structure of the molecule. Non-polar molecules have a higher capacity factor compared to polar molecules in Reverse phase chromatography. Polar molecules have a higher Capacity factor in normal phase chromatographic mode compared to non-polar molecules in Reverse phase chromatography.
• Type of the Mobile phase: Capacity factor depends upon the type of the solvent, buffer concentration, pH and ratio of the solvent.
• Chemistry of the stationary phase: The capacity factor depends upon the chemistry of the stationary phase. Non-polar compounds have a higher capacity factor in the non-polar column than in the polar column and vice versa (in Reverse phase chromatography)
• The particle size of the stationary phase: The capacity factor depends upon the particle size of the stationary phase. Lower particles have a higher capacity factor than the higher particle size
• Column length: Capacity factor is directly proportional to the column length

Acceptance criteria; Capacity factor

• K ≤1: Tells that the analyte quickly elutes from the column and there is no interaction of the analytes with the stationary phase
• K>1: Tells that the analyte interacts with the column and mobile phase and retains in the column.
• K=between 2 to 10: For a good chromatographic method K should be between 2 to 10.
• In certain conditions; values beyond 2 to 10 are also acceptable with proper scientific justification

Applications

• It is very helpful during method development while selecting the column, mobile phase
• It is also used as a system suitability parameter

Case study

A moderately acidic compound having a capacity factor of 1.5 in the C18 column using a mixture of water and acetonitrile in a ratio of 40:60 and a flow rate of 1.0 ml/minute. t_o of the column is 0.5 minutes. How more than 2 capacity factors can be achieved?

Discussion: Higher capacity factor can be achieved by following adjustment in the chromatographic condition:

• Use an acidic buffer in place of water since the molecule is acidic
• You may Increase the buffer composition and decrease the acetonitrile composition in the mobile phase
• You may decrease the flow rate of the mobile phase
• You may use C8 column in place of C18 column

Conclusion

Hope this post has cleared all your doubts related to capacity factor and now you can replicate effectively in chromatographic method development. That’s all about this article and for any suggestion or question related to this article write in the comment section. I will answer on a priority basis.

References

• Analytical Chemistry; Gary D. Christian

Abbreviations

• K = Capacity factor
• HPLC: High pressure liquid chromatography